Anticytokine Therapies in Rheumatoid Arthritis

Vibeke Strand, MD, Clinical Professor of Medicine, Stanford Medical
School, Edward Keystone, MD, Professor of Medicine, University of
Toronto, Arthritis Center of Excellence [UpToDate =A9 2000]

Cytokines mediate many aspects of immune function; they are produced by activated immune cells, and either enhance or inhibit the immune response. This card will review the different anticytokine therapies currently available and/or under evaluation for the management of patients with rheumatoid arthritis (RA).

T Cell Subsets, Cytokines, and Rheumatoid Arthritis
Before discussing the different anticytokine therapies in RA, it is
helpful to first briefly review current concepts concerning the
following issues:

The specific cytokines secreted by the different T helper subsets.
The influence these cells and molecules may have upon the development and perpetuation of RA. The different interventions proposed and recently introduced which attempt to ameliorate these processes.

T helper cells and the cytokines they produce can be categorized into two general subsets: Th1 and Th2.

Th1 lymphocytes act as inflammatory cells; they induce cell mediated responses which predominate in rheumatoid arthritis (RA), as well as acute allograft rejection, graft-versus-host disease, and reactive arthritis. In general, Th1 cells secrete the following proinflammatory mediators:[1]
Interleukin-1b (IL-1b)
Interferon gamma (IFNg)
Tumor necrosis factor alpha (TNFa).

Th2 lymphocytes stimulate antibody production by B cells and augment eosinophil responses. The activation of Th2 cells is associated with the development of chronic graft-versus-host disease, systemic lupus erythematosus, and scleroderma. Th2 cells secrete the following cytokines:

The cytokines generated by either Th1 or Th2 cells inhibit the cellular
function of the other phenotype.[2] Thus, IFNg inhibits Th2 cell
proliferation, while IL-10 inhibits Th1 production. However, the concept of Th1 or Th2 dominated immune responses appears too simplistic to consistently explain many of the responses observed following the administration of cytokine therapies. Other factors are also important.
These include the dose used, the frequency and route of administration, and the patient population being studied. Nevertheless, therapeutic strategies have been developed to selectively downregulate proinflammatory Th1 responses or upregulate antiinflammatory Th2 cytokine production in patients with RA.

Downregulation of Proinflammatory Cytokines
Interleukin-1b and TNFa are considered to be the major proinflammatory cytokines involved in the pathogenesis of RA and other chronic inflammatory diseases. When secreted by synovial macrophages, IL-1b and TNFa stimulate synovial cells to proliferate and synthesize collagenase, thereby degrading cartilage, stimulating bone resorption, and inhibiting proteoglycan synthesis.[3]

TNFa and IL-1b induce the expression of cellular adhesion molecules, thereby resulting in further inflammatory cell recruitment and release of additional cytokines. As an example, TNFa secretion stimulates release of IL-1b, IL-6, and arachidonic acid metabolites.[4] Using a transgenic mouse model, overproduction of TNFa resulted in the development of a chronic polyarthritis resembling RA.[5]

The significant role of IL-1b in the pathogenesis of RA is supported by the finding of increased levels of both IL-1b and its messenger RNA (mRNA) in the synovium and synovial fluid.[6] IL-1b has also been demonstrated in macrophages at the cartilage pannus junction and at sites of maximal cartilage and bone destruction.[7]

To downregulate or inhibit the effector functions of these cytokines in vivo, three general approaches have been used:
Cell surface receptor antagonist proteins [8]
Soluble receptor antagonists [9]
Antibodies to cytokines or their receptors.[10]

Cell Surface Receptor Antagonists
Recombinant cell surface receptor antagonists are biologically inactive proteins which compete with the cytokine for binding to the membrane receptor on the cell surface. To be effective, receptor antagonist proteins must bind greater than 90 percent of the receptors on the cell surface. Because large doses of the antagonist are required for such an effect, these proteins are less efficient despite a high specificity.

Soluble Receptor Antagonists
Soluble receptor antagonists are truncated forms of the cell surface
receptor devoid of the transmembrane and intracytoplasmic domains. However, they retain binding affinity comparable to the full length membrane-bound receptors. In contrast to cell surface receptor antagonist proteins, soluble receptor molecules bind free cytokine, inhibiting its ability to bind to cell surface receptors. A lower dose is required than for receptor antagonist proteins, but these molecules must remain within the circulation for longer periods of time to be effective.

To increase the half life of a soluble receptor in the circulation, a
variety of constructs incorporating structures such as the Fc receptor of human IgG1 have been employed.[11] However, these constructs may bind to cell surface Fc receptors present on antigen presenting cells, thereby increasing their immunogenicity. In addition, binding to a soluble receptor may prolong the half life of the cytokine in the circulation, thereby enhancing its effect.[12]

The third approach used to downregulate effector molecule function has been the use of anti-cytokine monoclonal antibodies (mAbs). Generally, these mAbs have a higher affinity for the target cytokine than the soluble receptor antagonist molecules and can bind to cell surface bound cytokine.

Upregulation of Inhibitory Cytokines
As previously mentioned, the products of Th1 and Th2 cells negatively regulate each other's functions.[2] As an example, IL-10 and IL-4 suppress Th1 responses in a synergistic manner.[13] IL-10 can also upregulate IL-1 receptor antagonist (IL-1ra) production. Therefore, IL-4 and IL-10 may be useful therapeutic agents in the treatment of RA.

A number of specific anticytokine therapies for the treatment of RA have been introduced into clinical practice. The remainder of this card will review the currently available therapies.

Tumor Necrosis Factor Alpha Antagonists
TNFa antagonists are approved for the treatment of RA by the United
States Food and Drug Administration. These include the synthesis of two soluble TNFa receptor fusion proteins, one targeting the p75 TNFa receptor and the other the p55 receptor, and anti-TNFa antibodies. The fusion proteins consist of genetic fusion of recombinant receptor to the Fc portion of IgG.

Soluble TNFa receptors (type II)
Etanercept (Enbrel=AE), a soluble p75 TNFa receptor fusion protein that binds TNF-alpha and has a longer half life than the native soluble receptor, has been approved by the Food and Drug Administration in the United States for the treatment of patients with moderate to severe RA who have responded inadequately to at least one disease modifying antirheumatic drug (DMARDS).

Several different studies have found that etanercept provides
substantial benefit to patients with RA: A three month, multicenter,
double-blind trial compared etanercept (0.25, 2, or 16 mg/m2),
administered subcutaneously twice weekly for three months, with
placebo.[14] Using ACR-20 (American College of Rheumatology 20 percent) criteria, there was significant improvement noted in the group assigned 16 mg of etanercept (75 versus 14 percent for placebo, P<0.001). The onset of this effect was rapid, and the benefit quickly reversed with treatment withdrawal.

In a six month multicenter, randomized, double-blind, placebo-controlled trial, 234 patients with active RA who had an inadequate response to DMARDs were randomized to twice weekly subcutaneous injections of etanercept, 10 or 25 mg, or placebo.[15] Using ACR-20 criteria,etanercept significantly reduced disease activity in the 25 mg dose treated group (59 versus 11 percent for placebo, P<0.001). In the 25 mg group, an ACR-50 response was achieved in 40 versus 5 percent for placebo (P<0.001). An ongoing long-term study has demonstrated the efficacy and safety of etanercept therapy continued for more than 18 months.[16]

To ascertain whether the addition of etanercept to methotrexate therapy would provide additional benefit to patients who had persistent RA despite methotrexate therapy, 89 patients were randomly assigned to receive etanercept 25 mg or placebo subcutaneously twice weekly while continuing to receive methotrexate.[17] At 24 weeks, patients receiving
etanercept plus methotrexate had significantly better outcomes according to all measures of disease activity. Seventy-one and 39 percent of patients receiving both drugs met ACR-20 and ACR-50 criteria respectively compared with 27 and 3 percent of those administered methotrexate alone (P<0.001).

Adverse effects
Etanercept is well tolerated. The most common adverse event has been injection site reactions, occurring in 37 percent of patients; all such reactions were reported as mild to moderate in severity.[18] They typically occurred during the first month of treatment and lasted for 3 to 5 days; rotating the site of injection usually ameliorated the problem.

Serious infections have been observed in 22 of 745 patients (2.9
percent). Recently, the label was updated to also report that six deaths have occurred because of infection among the estimated 25,000 patients treated with this agent.[19]

Overall, 16 percent of patients administered etanercept have developed non-neutralizing antibodies to the soluble receptor. In addition, 11 and 15 percent of etanercept treated patients have developed new positive ANAs and/or anti-ds DNA antibodies compared with 5 and 4 percent of those administered placebo, respectively. No cases of systemic lupus erythematosus or lymphoproliferative disorders have been reported.

Further study is required to accurately assess the proper role for
etanercept either as a single agent or in combination with other
DMARDs.[20] Etanercept is contraindicated in women who are pregnant or nursing, and patients who have an active infection.

Soluble TNFa Receptors (Type I)
Lenercept, a fusion protein that targets the soluble p55 receptor of TNF has also been studied in patients with RA. In a 12 week study of 247 patients with RA, lenercept was used alone and in combination with methotrexate.[21] Using the ACR-20 criteria, there was no statistical difference observed between the treatment groups (ACR-20 criteria achieved by 53 percent of patients receiving lenercept, 64 percent of patients receiving lenercept with methotrexate, and 50 percent of patients receiving methotrexate alone).

Side effects of lenercept were generally mild, consisting of injection
site reactions and mild infections primarily of the upper respiratory
tract. Although the development of an antibody response to lenercept was common, it was not associated with adverse effects and did not alter efficacy.[22] In addition, the development of newly positive antinuclear antibody responses and/or anti-ds DNA antibodies without the development of SLE was observed in 13 and 3 percent of treated patients, respectively.[23]

Since the benefit with lenercept did not exceed that from methotrexate administered alone, this agent was withdrawn from further clinical trials in patients with RA.

Chimeric Anti-TNFa Monoclonal Antibodies
Based upon encouraging data in the transgenic TNF murine model of arthritis and in collagen-induced arthritis in the mouse,[24] a chimeric (human/murine) IgG1 monoclonal antibody (mAb) to TNFa (cA2 or infliximab) was studied in an open label clinical trial in 20 patients with refractory RA. Following a single intravenous dose of infliximab, there was a marked improvement in systemic symptoms of malaise and fatigue which correlated with decreases in the levels of C-reactive protein.[25]

Similar benefits were observed in a placebo, randomized controlled trial in 73 patients with longstanding RA.[26] Using a Paulus 20 percent response at one month as the criterion for improvement, this target was achieved at 4 weeks in 79 percent of patients treated with high dose (10 mg/kg) of infliximab versus only 8 percent receiving placebo (P<0.0001). Over one-half of patients receiving high-dose infliximab attained a 50 percent Paulus response (P =3D 0.005).

These benefits were also confirmed in a study which randomized 428 patients being treated with methotrexate to one of five groups:
infliximab at 3 mg/kg administered either every month or every other
month, infliximab at 10 mg/kg also administered every month or every other month, or placebo.[27] All patients receiving active therapy were initiated with infliximab administered at zero, two and six weeks. At 30 weeks, a minimum ACR 20 response was observed in 50, 60, and 20 percent of the low dose, high dose, and placebo groups, respectively; ACR 50 responses were seen in 25 to 30, and 5 percent of the infliximab-treated and placebo groups, respectively. There was no statistical difference between the high and low dose, and monthly and every other month regimens. Benefits were principally observed within the first six weeks
of therapy. Given this evidence of efficacy, infliximab has been
approved by the Food and Drug Administration for use in patients with RA.

Development of Human Antichimeric Molecule Antibodies
The development of human anti-chimeric molecule antibody (HACA)
responses which recognize the murine component of infliximab is detected in 40 percent of patients when infliximab is used alone.[28] Whether these antibodies impair the clinical response is unknown.

In an effort to reduce the HACA responses and possibly prolong the
duration of clinical benefit, infliximab was administered at doses of 1,
3 and 10 mg/kg in combination with low dose (7.5 mg/week) methotrexate (MTX).[29] Although MTX administration appeared to have little effect on the peak clinical response, it prolonged the duration of responses in most cases. The development of a HACA response was inversely related to dose administered: 53 percent of patients receiving 1 mg/kg developed antibodies, compared with 21 percent of patients receiving 3 mg/kg and seven percent of those receiving 10 mg/kg. Concomitant methotrexate reduced the responses to 15, 7 and 0 percent, respectively, in these
three treatment groups.

Adverse Effects
Adverse events with this agent include headache, diarrhea, rash,
pharyngitis, rhinitis, cough, upper respiratory and urinary tract
infection.[26] These common infections were reported in 32 percent of infliximab treated patients compared with 21 percent of patients treated with placebo plus MTX. Infusion reactions (urticaria, pruritis and chills) caused 5 patients in the active treatment group to withdraw, two patients developed severe infections 9 and 15 weeks after receiving mAb treatment. Anti-double stranded DNA antibodies occurred in 7 patients (8 percent); one patient developed clinical manifestations of systemic lupus erythematosus.

In total, three cases of SLE and six cases of lymphoproliferative
disorders have been described following treatment with this agent, in patients with Crohn's disease and RA.[29-31] It is unclear whether these cases of lymphoproliferative disease reflect the propensity of patients with severe RA to develop lymphoma.[32-34] However, when compared to the frequency of malignancy with MTX, the incidence following treatment with biologic agents may be overestimated, as patients participating in clinical trials of experimental agents are subject to detailed and
prolonged surveillance.

It is also difficult to assess the true relationships related to the
development of a lymphoproliferative disorder, the duration and/or
severity of RA (or other underlying disease), and the forms, duration,
and strength of previous therapy (either with MTX or other
immunosuppressive agents).[35,36] As an example, a patient with Crohn's disease who developed lymphoma also had long-standing disease (more than 30 years) and was also taking azathioprine, both of which may increase the risk of lymphoma.[37]

This mAb was recently approved for the treatment of refractory Crohn's disease, including patients with fistula formation. Rapid and long lasting improvement has been observed in patients with this

Choice of Infliximab Versus Etanercept
Among the anticytokine therapies thus far studied, infliximab and
etanercept appear to be the most effective and extensively evaluated. The use of these biologic agents will likely be confined at present to patients with moderate to severe RA who are partial responders to optimal doses of currently accepted therapies such as MTX or to patients who are unable to tolerate MTX and have failed to respond to other disease modifying anti-rheumatic drugs (DMARDs). The high cost of these agents and parenteral administration will demand careful selection of

If such therapy is considered, the choice of one or the other agent will depend upon the patient, proven efficacy, and incidence and severity of adverse effects. As an example, use of the chimeric antibody infliximab was associated with the development of anti-chimeric antibodies, which lessened when used in combination with MTX. Since both infliximab and etanercept are likely to be used initially in conjunction with MTX, the difference in the incidence of antibodies may not be clinically important; however, if monotherapy is desired, etanercept may be the preferred choice of therapy at present.[20]

Only prolonged use of these agents will offer a true evaluation of their safety and efficacy. A small number of cases of lymphoproliferative disease and SLE have developed following treatment with the TNFa antagonists. The true significance of these numbers will only be ascertained following widespread clinical use.

Human Anti-TNFa mAb
A fully human recombinant IgG1 anti-TNFa mAb (D2E7) shows promise in an initial study in patients with RA. In a four week, double-blind, placebo controlled trial, single intravenous infusions of 0.5 to 10 mg/kg were administered to 120 patients with refractory RA.[41] Using a Disease Activity Score (DAS), improvement was achieved in 78 percent of patients receiving 10 mg/kg of D2E7 compared with 19 percent of patients receiving placebo infusions. Treatment was generally well tolerated.

Open label studies of repetitive intravenous infusions at approximately 2.5 week intervals revealed the maintenance of efficacy.[42] A double-blind, placebo controlled, randomized trial of weekly subcutaneous administration of D2E7 (0.5 mg/kg) in 24 patients with RA also demonstrated improvement.[43] Using ACR-20 criteria, a response rate of 67 percent at 12 weeks was observed in the active treatment group compared with a placebo response of 18 percent at 3 weeks. Given these promising findings, further trials are in progress.

Interleukin-1 Receptor Antagonist
The interleukin-1 (IL-1) receptor antagonist protein (IL-1ra) is a
naturally occurring glycoprotein inhibitor of IL-1 that binds the high
affinity cell surface IL-1 receptor but has no receptor activation
activity.[44] The agonist effects of IL-1 are therefore partially
regulated by IL-1ra.

IL-1ra is released primarily by monocytes and tissue macrophages.
Interleukin 1-ra inhibits prostaglandin production by synovial cells and chondrocytes and matrix metalloproteinase production by activated synovial cells and articular chondrocytes.[45,46]

The efficacy of IL-1ra in RA was studied in a double-blind,
placebo-controlled 24 week multicenter trial of 472 patients with active RA.[47] Patients were randomized to receive one of four treatments:
IL-1ra in a dose of 30, 75 or 150 mg/day (self-administered
subcutaneously), or placebo. Using ACR-20 criteria, there was
significant improvement in the high dose group (43 versus 27 percent for placebo). At 24 weeks, there was significant slowing of radiographic progression with active treatment as determined by serial Larsen scores.

Transient injection site reactions were the most frequent adverse
effects reported, resulting in a five percent withdrawal rate in the
high dose group. Serious infections developed in five patients in the
high dose group versus one individual administered placebo.

IL-1 Receptor Antagonist Gene Therapy
The onset of murine collagen induced arthritis was abrogated by
transfecting the knee synovium fibroblasts with the IL-1ra gene.[48]
Based on this and other encouraging animal model data,[49] gene therapy using retroviral constructs encoding human IL-1ra is underway in patients with RA.[50] The therapy involves removal of synovium from the joint of a patient awaiting total joint arthroplasty, transfecting it with the IL-1ra gene, and reimplanting it at the time of joint replacement.

In three patients, the procedure was well tolerated and expression of
the transgene was documented.[50] Further studies are currently in

IL-6 Antagonists
Interleukin-6 (IL-6) has both pro- and antiinflammatory activity,[52]
and increased synovial fluid levels correlate with disease activity in
RA.[53] Circulating levels of IL-6, but not TNFa or IL-1b, are elevated
in children with systemic juvenile RA.[54] In preclinical studies,
collagen-induced arthritis was ameliorated in monkeys using a humanized mAb to the IL-6 receptor (IL-6R).[55]

A murine anti IL-6 mAb was administered to patients with RA in a phase 1 open-label study.[56] Although a clinical benefit was observed,[56] administration of these antibodies can result in formation of immune complexes which markedly increase the half life of the cytokine.[57] These complexes, consisting of IL-6/IL-6R, behave as receptor agonists, binding IL-6 and prolonging its activity in the circulation.[58] This observation may limit the use of this approach clinically. To be effective, specific antagonists must bind the IL-6 receptor with greater avidity than IL-6, but should not induce activity.

In several animal models of autoimmunity, the administration of IL-4, an inhibitory cytokine, has ameliorated clinical disease.[59,60] However, preliminary studies in humans with RA have failed to demonstrate significant clinical benefit.

IL-10 levels are increased in serum and synovial fluids of RA patients, and IL-10 mRNA expression is increased in the peripheral blood mononuclear cells and synovial fluid cells of RA patients.[61,62] Addition of neutralizing monoclonal antibodies to IL-10 in RA cell cultures resulted in increased interferon, TNFa, IL-1b, and GM-CSF levels consistent with the concept that IL-10 is an endogenous inhibitor in the tissues of patients with RA.[63]

Because of these promising in vitro and in vivo findings, a phase I
multicenter, double-blind, placebo randomized controlled trial of daily, subcutaneously administered recombinant human IL-10 was initiated in patients with RA.[64] Trends toward improvement were observed, primarily with the 5 =B5g/kg dose. Adverse events included mild reversible dose dependent thrombocytopenia and anemia. Further studies, including the combination of IL-10 with MTX, are planned.

Gene therapy with IL-10 may also offer promise for the treatment of RA. The potential effectiveness of IL-10 was demonstrated in a study in which the gene for IL-10 was transferred into human synovial fibroblasts and subsequently implanted in SCID mice.[65] The expression of IL-10 in synovial fibroblasts decreased cartilage destruction compared with non-transfected animals. In addition, systemic IL-10 gene therapy consisting of intravenous injection of adenoviral vectors containing the gene has been found to prevent the development of collagen-induced arthritis in animals.[66-68]

Autoimmune Syndromes
Manipulation of the cytokine milieu, either by administering targeted
therapy or directly giving cytokines, may be associated with autoimmune phenomena. Autoimmune syndromes have been reported in patients treated with granulocyte colony stimulating factor (G-CSF), granulocyte monocyte stimulating factor (GM-CSF), interleukin 2 (IL-2), interferons (IFNs) alpha (a), beta (b), and gamma (g), and TNFa antagonists. As examples:[69-72]

G-CSF and GM-CSF therapy has been associated with the development and/or exacerbation of leukocytoclastic vasculitis, other inflammatory dermatologic conditions, and RA.

The administration of IFNg is associated with the development of
antinuclear antibodies in patients with RA, as well as the induction or
exacerbation of systemic lupus erythematosus (SLE) in those with RA and myeloproliferative disorders.[71]

Following treatment with IFNa, an increased incidence of thyroiditis,
RA, and SLE has been reported in patients with malignancy or chronic hepatitis C infection.[72]

As previously mentioned, the development of anti DNA antibodies has been reported in as many as 15 percent of patients receiving treatment with the TNFa antagonists (soluble receptors and monoclonal antibodies). Thus far, the number of cases of treatment related-SLE has remained small, and the autoantibodies do not persist once therapy is withdrawn.

At present, only the therapies directed against the activity of TNFa
have been proven to be an effective treatment modality in active RA.
Combination therapy in patients with active disease, despite long term administration of methotrexate, offers additional benefit. Clinical
trials are underway to assess whether treatment will significantly delay radiographic progression of the disease.

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